RNA acts as information carriers, catalysts, and regulators in essential biological processes, and understanding RNA structure and function is intimately linked to combating diseases. The long-term objectives of this project is to advance our understanding of the RNA structure/function relationship via studying structure, conformational dynamics, and mechanism of function, in the phi29 packaging RNA (pRNA) that plays essential roles in the most powerful biological motor known today - the DNA packaging motor of the phi29 bacteriophage. The specific aims involve a parallel development of the site-directed spin labeling (SDSL) technique for studying RNA structure and dynamics, and application of SDSL and other methods to study pRNA structure and interaction. SDSL utilizes a nitroxide moiety attached to specific sites to obtain structural and dynamic information of the parent molecule, and has recently been shown to be able to provide unique information in RNA. Proposed RNA SDSL developments, which are driven by specific questions in pRNA studies, include: (I) establishing long-range distance measurements in nucleic acids using nitroxides attached to arbitrary sequences;and (ii) establishing the first empirical RNA SDSL line shape library for deducing RNA structural information from singly labeled nitroxides. These developments will find applications in packaging RNA studies. Specific aims includes: (I) studying structure and conformational changes in the pRNA using a combination of SDSL, NMR, and computational modeling;and (ii) mapping pRNA/protein and pRNA/pRNA interfaces in the motor via analyzing pRNA functional groups. The proposed studies will reveal information on packaging RNA structure and interaction that will aid in understanding the mechanism of phi29 packaging. It is believed that phi29 shares the same DNA packaging mechanism with many other double stranded DNA viruses, including a number of human pathogens. Understanding phi29 packaging motor function will aid in developing pharmacological approaches to combat these viruses.